Bovine Muscle Research Articles

Myostatin Mutation Enhances Bovine Myogenic Differentiation through PI3K/AKT/mTOR Signalling via Removing DNA Methylation of RACK1.

Myostatin (MSTN) is a negative regulator of skeletal muscle development and plays an important role in muscle development. Fluctuations in gene expression influenced by DNA methylation are critical for homeostatic responses in muscle. However, little is known about the mechanisms underlying this fluctuation regulation and myogenic differentiation of skeletal muscle. Here we report a genome-wide analysis of DNA methylation dynamics in bovine skeletal muscle myogenesis after myostatin editing. We show that, after myostatin editing, an increase in TETs (DNA demethylases) and a concomitant increase in the receptor for activated C kinase 1 (RACK1) control the myogenic development of skeletal muscle. Interestingly, enhancement of PI3K/AKT/mTOR signaling by RACK1 appears to be an essential driver of myogenic differentiation, as it was associated with an increase in myogenic differentiation marker factors (MyHC and MyoG) during muscle differentiation. Overall, our results suggest that loss of myostatin promotes the myogenic differentiation response in skeletal muscle by decreasing DNA methylation of RACK1.

Relationships between the abundance of 29 proteins and several meat or carcass quality traits in two bovine muscles revealed by a combination of univariate and multivariate analyses

We aimed to evaluate the relationships between meat or carcass properties and the abundance of 29 proteins quantified in two muscles, Longissimus thoracis and Rectus abdominis, of Rouge des Prés cows. The relative abundance of the proteins was evaluated using a high throughput immunological method: the Reverse Phase Protein array. A combination of univariate and multivariate analyses has shown that small HSPs (CRYAB, HSPB6), fast glycolytic metabolic and structural proteins (MYH1, ENO3, ENO1, TPI1) when assayed both in RA and LT, were related to meat tenderness, marbling, ultimate pH, as well as carcass fat-to-lean ratio or conformation score. In addition to some small HSP, ALDH1A1 and TRIM72 contributed to the molecular signature of muscular and carcass adiposity. MYH1 and HSPA1A were among the top proteins related to carcass traits. We thus shortened the list to 10 putative biomarkers to be considered in future tools to manage both meat and carcass properties. SignificanceIn three aspects this manuscript is notable. First, this is the first proteomics study that aims to evaluate putative biomarkers of both meat and carcass qualities that are of economic importance for the beef industry. Second, the relationship between the abundance of proteins and the carcass or meat traits were evaluated by a combination of univariate and multivariate analyses on 48 cows that are representative of the biological variability of the traits. Third, we provide a short list of ten proteins to be tested in a larger population to feed the pipeline of biomarker discovery.

Cis-eQTL Analysis and Functional Validation of Candidate Genes for Carcass Yield Traits in Beef Cattle.

Carcass yield traits are of considerable economic importance for farm animals, which act as a major contributor to the world’s food supply. Genome-wide association studies (GWASs) have identified many genetic variants associated with carcass yield traits in beef cattle. However, their functions are not effectively illustrated. In this study, we performed an integrative analysis of gene-based GWAS with expression quantitative trait locus (eQTL) analysis to detect candidate genes for carcass yield traits and validate their effects on bovine skeletal muscle satellite cells (BSCs). The gene-based GWAS and cis-eQTL analysis revealed 1780 GWAS and 1538 cis-expression genes. Among them, we identified 153 shared genes that may play important roles in carcass yield traits. Notably, the identified cis-eQTLs of PON3 and PRIM2 were significantly (p < 0.001) enriched in previous GWAS loci for carcass traits. Furthermore, overexpression of PON3 and PRIM2 promoted the BSCs’ proliferation, increased the expression of MYOD and downregulated the expression of MYOG, which indicated that these genes may inhibit myogenic differentiation. In contrast, PON3 and PRIM2 were significantly downregulated during the differentiation of BSCs. These findings suggested that PON3 and PRIM2 may promote the proliferation of BSCs and inhibit them in the pre-differentiation stage. Our results further contribute to the understanding of the molecular mechanisms of carcass yield traits in beef cattle.

Acoustic and Thermal Characterization of Therapeutic Ultrasonic Langevin Transducers under Continuous- and Pulsed Wave Excitations.

We previously conducted an empirical study on Langevin type transducers in medical use by examining the heat effect on porcine tissue. For maximum acoustic output, the transducer was activated by a continuous sinusoidal wave. In this work, pulsed waves with various duty factors were applied to our transducer model in order to examine their effect on functionality. Acoustic power, electro-acoustic conversion efficiency, acoustic pressure, thermal effect on porcine tissue and bovine muscle, and heat generation in the transducer were investigated under various input conditions. For example, the results of applying a continuous wave of 200 VPP and a pulse wave of 70% duty factor with the same amplitude to the transducer were compared. It was found that continuous waves generated 9.79 W of acoustic power, 6.40% energy efficiency, and 24.84 kPa acoustic pressure. In pulsed excitation, the corresponding values were 9.04 W, 8.44%, and 24.7 kPa, respectively. The maximum temperature increases in bovine muscle are reported to be 83.0 °C and 89.5 °C for each waveform, whereas these values were 102.5 °C and 84.5 °C in fatty porcine tissue. Moreover, the heat generation around the transducer was monitored under continuous and pulsed modes and was found to be 51.3 °C and 50.4 °C. This shows that pulsed excitation gives rise to less thermal influence on the transducer. As a result, it is demonstrated that a transducer triggered by pulsed waves improves the energy efficiency and provides sufficient thermal impact on biological tissues by selecting proper electrical excitation types.

Transcriptome-Wide Study of mRNAs and lncRNAs Modified by m6A RNA Methylation in the Longissimus Dorsi Muscle Development of Cattle-Yak.

Cattle-yak is a hybrid F1 generation of cattle and yak, which has a history of more than 3000 years and has shown better production performance and higher economic benefits than those of yaks. However, up to now, there has been no study on the transcriptome-wide m6A methylation profile of bovine skeletal muscle and its potential biological function during muscle development. Here, we observed significant changes in the expression levels of muscle-related marker genes and methylation-related enzymes during the development of cattle-yak, and the overall m6A content in the Longissimus dorsi muscle of 18-month-old cattle-yak decreased significantly. A total of 36,602 peaks, 11,223 genes and 8388 lncRNAs were identified in the two groups, including 2989 differential peaks (427 up-regulated peaks and 2562 down-regulated peaks), 1457 differentially expressed genes (833 up-regulated genes and 624 down-regulated genes) and 857 differentially expressed lncRNAs (293 up-regulated lncRNAs and 564 down-regulated lncRNAs). GO and KEGG analysis revealed that they were significantly enriched in some muscle-related pathways (Wnt signaling pathway and MAPK signaling pathway) and high-altitude adaptation-related pathway (HIF-1 signaling pathway). Moreover, m6A abundance was positively correlated with gene expression levels, while it was negatively correlated with lncRNA expression levels. This indicates that m6A modification played an important role in the Longissimus dorsi muscle development of cattle-yak; however, the regulation mechanism of m6A-modified mRNA and lncRNA may be different. This study was the first report of transcriptome-wide m6A-modified mRNAs and lncRNAs atlas in the Longissimus dorsi muscle development of cattle-yak, one which will provide new perspectives for genetic improvement in bovines.

Fast and Sensitive Analysis of Short- and Long-Chain Perfluoroalkyl Substances in Foods of Animal Origin.

The availability of sensitive analytical methods to detect per- and polyfluoroalkyl substances (PFASs) in food of animal origin is fundamental for monitoring programs to collect data useful for improving risk assessment strategies. The present study aimed to develop and validate a fast and sensitive method for determining short and long-chain PFASs in meat (bovine, fish, and swine muscle), bovine liver, hen eggs, and cow's milk to be easily applicable in routine analysis of food. A QuEChERS extraction and clean-up method in combination with liquid chromatography coupled to mass spectrometry (LC-MSMS) were used. The method resulted in good linearity (Pearson's R &gt; 0.99), low limits of detection (7.78-16.35 ng/kg, 8.26-34.01 ng/kg, 6.70-33.65 ng/kg, and 5.92-19.07 ng/kg for milk, liver, egg, and muscle, respectively), and appropriate limits of quantification (50 ng/kg for all compounds except for GenX and C6O4, where the limits of quantification were 100 ng/kg). Trueness and precision for all the tested levels met the acceptability criteria of 80-120% and ≤20%, respectively, regardless of the analyzed matrix. As to measurement uncertainty, it was &lt;50% for all compound/matrix combinations. These results demonstrate the selectivity and sensitivity of the method for simultaneous trace detection and quantification of 14 PFASs in foods of animal origin, verified through the analysis of 63 food samples.

A SNP of the COX4I2 gene associated with environmental adaptation in Chinese cattle

COX4I2 is an isoform of cytochrome C oxidase subunit IV (COX4), which plays an important role in mitochondrial oxidative phosphorylation. This gene affects heat production and thus affects thermoregulatory capacity in mammals. A splice region variant (rs109072064, NC_037340.1:g.61202988C > T) was identified in COX4I2 by using Ensembl, which transforms the amino acid arginine into cysteine in XP_005214921.1. In this study, we sought to determine the relationship between the mutant locus and the environment in which the cattle are located. We verified that mRNA (XM_005214864.4), which translated XP_005214921.1, is expressed in bovine muscle, fat, heart, liver, kidney, lung and testis tissues. The g.61202988C > T variant was then genotyped in 569 individuals of 34 cattle breeds. Compared with the CC genotype, southern cattle carried more the CT and TT genotypes. Furthermore, the association results carried out that the frequencies of genotypes (CC, CT, TT) and the value of climate parameters (mean annual temperature (T), relative humidity (RH) and temperature humidity index (THI)) were significantly correlated (P < 0.01). Hence, we speculated that the g.61202988C > T variant of COX4I2 gene was associated with the environmental adaptation trait in Chinese cattle and the locus may be considered as a molecular marker for Chinese cattle breeding.

Effects of transcription factor SIX2 gene on the proliferation of bovine skeletal muscle satellite cells

Objective: To investigate the effect of SIX2 gene on the proliferation of bovine skeletal muscle satellite cells. Methods: Bovine skeletal muscle satellite cells were used as experimental materials, and the expression of SIX2 gene in bovine skeletal muscle satellite cells was detected by real-time quantitative PCR at 24 h, 48 h, and 72 h of proliferation. The SIX2 gene overexpression vector was constructed by homologous recombination. The SIX2 gene overexpression plasmid and the control empty plasmid were transfected into bovine skeletal muscle satellite cells, and each group had three complex Wells. The cell viability was detected by MTT assay at 24 h, 48 h and 72 h after transfection. At 48 h after transfection, the cell cycle was detected by flow cytometry, and the expressions of cell proliferation marker genes were detected by real-time quantitative PCR (qRT-PCR) and Western blot. Results: With the proliferation of bovine skeletal muscle satellite cells, the expression of SIX2 mRNA was increased. Compared with the control group, the expressions of SIX2 mRNA and protein in the SIX2 gene overexpression plasmid group were increased by 18 and 2.6 times, respectively (P＜0.01). The cell viability of the SIX2 gene overexpression plasmid group was increased (P＜0.01), the proportion of G1 cells was decreased by 24.6%, and the proportion of S phase and G2 phase cells was increased by 20.3% and 4.31%, respectively (P＜0.01). The mRNA and protein expressions of Pax7 gene were increased by 15.84 and 1.22 times, respectively, and the mRNA and protein expressions of proliferation marker genes PCNA and CCNB1 were increased by 4.82, 2.23,1.55 and 1.46 times, respectively (P＜0.01). Conclusion: Overexpression of SIX2 gene promotes the proliferation of bovine skeletal muscle satellite cells.

Physicochemical Properties and Young Adult Consumer Preference of Dry-Aged Beef after High-Intensity Ultrasonication

This study aimed to determine the effect of high-intensity ultrasound (HIU. F = 37 kHz, I = 28W/cm2, bath for 30 min, 5 °C) on physicochemical characteristics and sensorial preference of seven aged (23 d ageing) bovine muscles (L. dorsi lumborum, L. dorsi thoracis, Psoas major, Semimembranosus, Biceps femoris, Rectus femoris, and Gluteus medius). Muscles were randomly distributed in two treatments: with and without ultrasonication. Colour (L*, a*, b* and C*), water-holding capacity (WHC), and shear force (N) were determined before and after simulated retail display (SRD) in modified atmosphere packing (MAP; 75% O2: 25% CO2, 3 °C, 13 h led light exposition) for 5 d. Sensorial toughness was also evaluated at the end of the SRD. Ultrasonication slightly reduces 6–9% WHC of beef. HIU did not affect (p ˃ 0.05) water loss, meat colour, shear force and sensorial toughness of the meat. The Semimembranosus was the toughest muscle. Ultrasonication of 23-day-aged beef did not show improvements on quality characteristics, and despite minor changes in water loss and slight increase in shear force, consumers did not detect differences.

Effects of Marination with Different Salt Concentrations on Some Quality Criteria of Beef Meat

In the present study, the effect of different salt concentrations on some quality properties such as the total number of coliform bacteria, texture, cooking loss, total salt content, metmyoglobin, pH, the water activity of steaks prepared from longissimus dorsi muscle of fresh beef was investigated. For this purpose, steaks prepared from longissimus dorsi muscle were dipped in marination brine in four different concentrations; 0% (control group), 1.5% NaCl, 2% NaCl and 2.5% NaCl, and marinated for 24 hours at +4 °C. The initial crude protein, crude fat, pH, moisture, and ash contents of beef were determined as 20.50%, 5.80%, 6.13, 72.63%, 1.07%, respectively. In the cooked meat samples, the hardness, chewiness and gumminess of the samples marinated with 1.5% and 2% NaCl was found to be higher than the control group. Metmyoglobin content of the meat samples marinated with NaCl is lower than the control group. The results revealed that the total number of coliform bacteria decreased drastically in parallel with the increase in salt concentration. As a result, the changes in the quality of the bovine longissimus dorsi muscle with different salt concentrations and marination were investigated, and positive effects on the textural properties and coliform bacterial load of the meat were observed.

Muscle fiber type-specific proteome distribution and protease activity in relation to proteolysis trends in beef striploin (M. longissimus lumborum) and tenderloin (M. psoas major)

The muscle fiber type and proteome distributions, protease activity, and protein degradation in bovine M. longissimus lumborum (beef striploin; LL) and M. psoas major (beef tenderloin; PM) were assessed to study muscle type-specific proteolysis. Bovine LL and PM muscle pieces (3 cm thickness) were stored in a refrigerator for 14 days and protein degradation and protease activity were evaluated at 1, 7, and 14 days of storage. Bovine LL included more fast-twitch glycolytic (type IIX) fibers than PM, whereas PM had a higher composition of slow-twitch oxidative (type I) fibers than LL (P < 0.05). Proteome distribution between these two muscles showed muscle fiber type-specificities: LL showed high intensities of proteins responsible for glycolytic metabolism, while PM showed large distribution of mitochondrial proteins. The activity of proteases, mainly calpain, showed different tendencies between the two muscles during 14 days of cold storage, which resulted in different timing of excessive myofibrillar protein breakdown (myosin heavy chains 1 and 2) between the two muscles. These results provided a detailed understanding of the differences in proteolysis by muscle type resulting in proteolysis-induced changes in meat quality during storage.

MiR-183/96/182 Cluster Regulates the Development of Bovine Myoblasts through Targeting FoxO1.

Simple SummaryIn this work, we identified that the miR-183/96/182 cluster was highly expressed in bovine embryonic muscle; meanwhile, it widely existed in other organizations. Functional assays indicated that the miR-183/96/182 cluster targets the FoxO1 gene to regulate the proliferation and differentiation of bovine myoblasts.Muscle development is an important factor affecting meat yield and quality and is coordinated by a variety of the myogenic genes and signaling pathways. Recent studies reported that miRNA, a class of highly conserved small noncoding RNA, is actively involved in regulating muscle development, but many miRNAs still need to be further explored. Here, we identified that the miR-183/96/182 cluster exhibited higher expression in bovine embryonic muscle; meanwhile, it widely existed in other organizations. Functionally, the results of the RT-qPCR, EdU, CCK8 and immunofluorescence assays demonstrated that the miR-183/96/182 cluster promoted proliferation and differentiation of bovine myoblast. Next, we found that the miR-183/96/182 cluster targeted FoxO1 and restrained its expression. Meanwhile, the expression of FoxO1 had a negative correlation with the expression of the miR-183/96/182 cluster during myoblast differentiation. In a word, our findings indicated that the miR-183/96/182 cluster serves as a positive regulator in the proliferation and differentiation of bovine myoblasts through suppressing the expression of FoxO1.

Internal calibration for opportunistic computed tomography muscle density analysis.

Muscle weakness can lead to reduced physical function and quality of life. Computed tomography (CT) can be used to assess muscle health through measures of muscle cross-sectional area and density loss associated with fat infiltration. However, there are limited opportunities to measure muscle density in clinically acquired CT scans because a density calibration phantom, allowing for the conversion of CT Hounsfield units into density, is typically not included within the field-of-view. For bone density analysis, internal density calibration methods use regions of interest within the scan field-of-view to derive the relationship between Hounsfield units and bone density, but these methods have yet to be adapted for muscle density analysis. The objective of this study was to design and validate a CT internal calibration method for muscle density analysis. We CT scanned 10 bovine muscle samples using two scan protocols and five scan positions within the scanner bore. The scans were calibrated using internal calibration and a reference phantom. We tested combinations of internal calibration regions of interest (e.g., air, blood, bone, muscle, adipose). We found that the internal calibration method using two regions of interest, air and adipose or blood, yielded accurate muscle density values (< 1% error) when compared with the reference phantom. The muscle density values derived from the internal and reference phantom calibration methods were highly correlated (R2 > 0.99). The coefficient of variation for muscle density across two scan protocols and five scan positions was significantly lower for internal calibration (mean = 0.33%) than for Hounsfield units (mean = 6.52%). There was no difference between coefficient of variation for the internal calibration and reference phantom methods. We have developed an internal calibration method to produce accurate and reliable muscle density measures from opportunistic computed tomography images without the need for calibration phantoms.

PFN1 Inhibits Myogenesis of Bovine Myoblast Cells via Cdc42-PAK/JNK.

Myoblast differentiation is essential for the formation of skeletal muscle myofibers. Profilin1 (Pfn1) has been identified as an actin-associated protein, and has been shown to be critically important to cellular function. Our previous study found that PFN1 may inhibit the differentiation of bovine skeletal muscle satellite cells, but the underlying mechanism is not known. Here, we confirmed that PFN1 negatively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Immunoprecipitation assay combined with mass spectrometry showed that Cdc42 was a binding protein of PFN1. Cdc42 could be activated by PFN1 and could inhibit the myogenic differentiation like PFN1. Mechanistically, activated Cdc42 increased the phosphorylation level of p2l-activated kinase (PAK), which further activated the phosphorylation activity of c-Jun N-terminal kinase (JNK), whereas PAK and JNK are inhibitors of myogenic differentiation. Taken together, our results reveal that PFN1 is a repressor of bovine myogenic differentiation, and provide the regulatory mechanism.

H2O2-induced oxidative stress improves meat tenderness by accelerating glycolysis via hypoxia-inducible factor-1α signaling pathway in postmortem bovine muscle.

Reactive oxygen species (ROS) affect meat quality through multiple biochemical pathways. To investigate the effect of ROS on postmortem glycolysis and tenderness of bovine muscle, ROS content, glycolytic potential, glycolysis rate-limiting enzyme activities, expression of hypoxia-inducible factor-1α (HIF-1α), phosphatidylinositol 3-kinase (PI3K), serine-threonine kinase (AKT), phosphorylated AKT (p-AKT), and tenderness were determined in the H2O2 group and control group. Results showed that the H2O2 group exhibited significantly higher ROS content within 48h, coupled with increased glycolytic potential, pH decline, hexokinase (HK), and phosphofructokinase activities (PFK) early postmortem. These were attributed to ROS-induced PI3K/AKT signaling pathway activation and resultant HIF-1α accumulation. Moreover, shear force in the H2O2 group reached the peak 12h earlier and decreased obviously after 24h, accompanied by a significantly higher myofibril fragmentation index (MFI). These findings suggested that ROS drive HIF-1α accumulation by activating PI3K/AKT signaling pathway, thereby accelerating glycolysis and tenderization of postmortem bovine muscle.

Interaction of MyoD and MyoG with Myoz2 gene in bovine myoblast differentiation

This study aims to explore the functional role of Myoz2 in myoblast differentiation, and elucidate the potential factors interact with Myoz2 in promoter transcriptional regulation. The temporal-spatial expression results showed that the bovine Myoz2 gene was highest expressed in longissimus dorsi, and in individual growth stages and myoblast differentiation stages. Knockdown of Myoz2 inhibited the differentiation of myoblast, and negative effect of MyoD, MyoG, MyH and MEF2A expression on mRNA levels. Subsequently, the promoter region of bovine Myoz2 gene with 1.7 Kb sequence was extracted, and then it was set as eight series of deleted fragments, which were ligated into pGL3-basic to detect core promoter regions of Myoz2 gene in myoblasts and myotubes. Transcription factors MyoD and MyoG were identified as important cis-acting elements in the core promoter region (−159/+1). Also, it was highly conserved in different species based on dual-luciferase analysis and multiple sequence alignment analysis, respectively. Furthermore, a chromatin immunoprecipitation (ChIP) analysis combined with site-directed mutation and siRNA interference and overexpression confirmed that the combination of MyoD and MyoG occurred in region −159/+1, and played an important role in the regulation of bovine Myoz2 gene. These findings explored the regulatory network mechanism of Myoz2 gene during the development of bovine skeletal muscle.

209 A Single-Cell Atlas of Bovine Skeletal Muscle Tissue Reveals Mechanisms Underlying Differences in Quality between Wagyu and Brahman Beef

Abstract Wagyu cattle are renowned for their premium beef characterized by abundant intramuscular fat (IMF). In contrast, Brahman, a popular breed in the southern coastal area of the US, generally produces tough meat with scarce IMF. However, the mechanisms underlying the differential meat quality between the 2 breeds are still largely unknown. Here, using single-cell RNAseq (scRNAseq), we report a single-cell atlas of bovine skeletal muscle tissue. Multiple fibro/adipogenic progenitor (FAP) subpopulations were identified, including an endomysial adipogenic subpopulation and a perimysial fibrogenic subpopulation. Further analysis comparing individual FAP subpopulations between Wagyu and Brahman identified stronger expression of pro-adipogenic genes in Wagyu adipogenic FAP subpopulation and higher expression of pro-fibrogenic genes in Brahman fibrogenic FAP subpopulation. Overexpression of CFD, a gene upregulated in Wagyu adipogenic FAPs, enhanced the adipogenic efficiency of FAPs. Moreover, the expression of CFD in FAPs was positively correlated with the IMF content. Interestingly, the expression of CFD was not identified in FAPs of mouse models of intramuscular adipogenesis, suggesting that CFD is a cattle-specific regulator of IMF formation. Cell-cell communication analysis reveals significant differences between CFD+ FAPs and CFD- FAPs in their interactions with other cell types, further suggesting an important role of CFD+ FAPs in bovine skeletal muscle growth and development.

4-Oxo-2-nonenal (4-ONE)-Induced Degradation of Bovine Skeletal Muscle Proteins.

Lipids are an important component of meat, as they provide desirable sensory characteristics and nutritional benefits. However, lipids are susceptible to degradation through oxidation and produce toxic oxidative byproducts. 4-Oxo-2-nonenal (4-ONE) is an oxidative byproduct that is highly reactive and cytotoxic. In this study, we investigated the influence of 4-ONE-induced protein degradation on fresh and gastric digested bovine skeletal muscle proteins. The results indicated that 4-ONE naturally forms in fresh muscle proteins. We report here for the first time that 4-ONE causes severe degradation of bovine skeletal muscle proteins. An SDS-PAGE gel analysis showed evidence that the skeletal muscle proteins attenuated over the incubation time, as the density of the protein bands faded significantly after 120 h. Additionally, protein and band density analyses showed a significant decrease in protein abundance and band densities throughout the incubation time. This study revealed that the lipid oxidation byproduct, 4-oxo-2-nonenal (4-ONE) is responsible for causing skeletal muscle protein degradation. Future studies should assess the bioprotective role of antioxidants and other food ingredients for their potential to prevent the formation and/or detoxification of 4-ONE in meat.

PSV-A-10 Single-Cell Atlas of Bovine Skeletal Muscle Identifies Mechanisms Regulating Intramuscular Adipogenesis and Fibrogenesis

Abstract Human and mouse studies have shown that fibro/adipogenic progenitors (FAPs) are a major source of intramuscular fat (IMF) and extracellular matrix (ECM) proteins. IMF and ECM proteins directly influence the palatability of beef, suggesting an essential role of FAPs in beef quality determination, which is still largely unexplored. We performed single-cell RNAseq (scRNAseq) using cells isolated from full blood Wagyu and Brahman cattle and Wagyu/Brahman cross cattle, which identified 21 cell clusters representing FAPs, several endothelial cell types, vascular smooths muscle cells, satellite cells, muscle fibers, and multiple immune cell types. More abundant FAPs were identified in the muscle of Brahman cattle, while a larger number of endothelial cells were identified in the muscle of Wagyu cattle. Further analysis of FAPs identified multiple FAP subpopulations with distinct gene expression profiles and anatomic locations. GSEA analysis revealed adipogenic and fibrogenic FAP subpopulations. A comparison of FAP subpopulations among different breeds showed higher complement system activity in the adipogenic FAP subpopulation of Wagyu cattle. Forced activation of the complement system in FAPs enhanced their adipogenic efficiency in vitro. In addition, cell-cell communication analysis identified active interactions between FAPs and other cell types through direct contact and secreted factors, many of which may affect FAP activities. In conclusion, our study revealed the single-cell atlas of bovine skeletal muscle and identified mechanisms regulating bovine intramuscular adipogenesis and fibrogenesis.

Engineered marble-like bovine fat tissue for cultured meat

Cultured meat can provide a sustainable and more ethical alternative to conventional meat. Most of the research in this field has been focused on developing muscle tissue, as it is the main component of meat products, while very few studies address cultured fat tissue, an essential component in the human diet and determinant of meat quality, flavor, juiciness, and tenderness. Here, we engineered bovine fat tissue for cultured meat and incorporated it within engineered bovine muscle tissue. Mesenchymal stem cells (MSCs) were derived from bovine adipose tissue and exhibited the typical phenotypic profile of adipose-derived MSCs. MSC adipogenic differentiation and maturation within alginate-based three-dimensional constructs were optimized to yield a fat-rich edible engineered tissue. Subsequently, a marble-like construct, composed of engineered bovine adipose and muscle tissues, was fabricated, mimicking inter- and intra-muscular fat structures.